Study On The Function And Mechanism Of PUMA And Bcl-X_L In Mitophagy

Colorectal cancer is a very common type of digestive tract malignant tumor and the third most common cancer in the world.Treatment modalities for colorectal cancer,most of them are chemoradiotherapy,surgical resection,targeted therapy as well as comprehensive treatment modalities.In addition,in the process of treatment,the elimination of tumor cells is mostly carried out by inducing cell death,in which autophagy or mitophagy may also exist.However,because the role of autophagy or mitophagy in colorectal cancer is complex and unclear,it has research interest.As key regulators of apoptotic pathways in tumor cells,PUMA and Bcl-X_L are widely studied,and some experiments have also found that they are associated with mitophagy.However,in colorectal cancer,whether they affect mitophagy and how mitophagy is regulated remain unclear.In this study,we explored the interaction among PUMA,ULK1,Beclin 1,and Bcl-X_L in different subcellular localizations,and their functions in autophagy and mitophagy respectively.We found that PUMA inhibited autophagy by forming a complex with ULK1 and Beclin1 in the cytoplasm.Therefore,we constructed six puma deletion mutants by considering the known full-length sequence of PUMA along with the corresponding function to further investigate which site is responsible for the interaction’s function and,on the basis of this,we found that the BH3 domain showed indispensable importance in the inhibition of autophagy by PUMA,through which PUMA could interact with ULK1 and play a role in inhibiting autophagy.By contrast,inducible autophagy was able to significantly degrade PUMA protein.More interestingly,when PUMA moved to mitochondria and formed a complex with ULK1and Bcl-X_L,they played total opposite roles,to promote mitophagy.During this process,Ser96 of PUMA was indispensable although all of the six mutants still could translocate to mitochondria.In addition,only over-expressed PUMA or Bcl-X_L induced the occurrence of mitophagy obviously,in which Drp1 may be involved,and the Real-Time detection of the subcellular localization of lysosome and mitochondria shows that increasing fusion was generated between them.Finally,protective autophagy or mitophagy was induced by targeted small molecular inhibitors during cancer therapies.In conclusion,our findings identified a novel functions and molecular mechanisms of PUMA and Bcl-X_L in autophagy and mitophagy,which supplied theoretical basis for clinical cancer therapy and other diseases.